Meulenbelt IM, Bhutani N, den Hollander W, Gay S, Oppermann U, Reynard LN, Skelton AJ, Young ...

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Meulenbelt IM, Bhutani N, den Hollander W, Gay S, Oppermann U, Reynard
LN, Skelton AJ, Young DA, Beier F, Loughlin J.
The first international workshop on the epigenetics of osteoarthritis.
Connective Tissue Research (2016)
DOI: http://dx.doi.org/10.3109/03008207.2016.1168409

Copyright:
© 2016 Ingrid M. Meulenbelt, Nidhi Bhutani, Wouter den Hollander, Steffen Gay, Udo Oppermann,
Louise N. Reynard, Andrew J. Skelton, David A. Young, Frank Beier, and John Loughlin. Published with
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Connective Tissue Research
ISSN: 0300-8207 (Print) 1607-8438 (Online) Journal homepage: http://www.tandfonline.com/loi/icts20
The first international workshop on the
epigenetics of osteoarthritis
Ingrid M. Meulenbelt, Nidhi Bhutani, Wouter den Hollander, Steffen Gay,
Udo Oppermann, Louise N. Reynard, Andrew J. Skelton, David A. Young,
Frank Beier & John Loughlin
To cite this article: Ingrid M. Meulenbelt, Nidhi Bhutani, Wouter den Hollander, Steffen
Gay, Udo Oppermann, Louise N. Reynard, Andrew J. Skelton, David A. Young, Frank Beier &
John Loughlin (2016): The first international workshop on the epigenetics of osteoarthritis,
Connective Tissue Research, DOI: 10.3109/03008207.2016.1168409
To link to this article: http://dx.doi.org/10.3109/03008207.2016.1168409
© 2016 Ingrid M. Meulenbelt, Nidhi Bhutani,
Wouter den Hollander, Steffen Gay, Udo
Oppermann, Louise N. Reynard, Andrew J.
Skelton, David A. Young, Frank Beier, and
JAochcne pLtoeudg ahulitnh. oPru vbelirsshioend pwoitshte ldic eonnslien eb:y 30
TMaayrlo 2r0 &1 6F.rancis
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CONNECTIVETISSUERESEARCH
http://dx.doi.org/10.3109/03008207.2016.1168409
MEETING REPORT
The first international workshop on the epigenetics of osteoarthritis
Ingrid M. Meulenbelta, Nidhi Bhutanib, Wouter den Hollandera, Steffen Gayc, Udo Oppermannd,e,
Louise N. Reynardf, Andrew J. Skeltonf,g, David A. Youngf, Frank Beierh and John Loughlinf
aDepartmentofMedicalStatisticsandBioinformatics,SectionofMolecular Epidemiology,LeidenUniversityMedicalCenter,Leiden,
TheNetherlands;bDepartmentofOrthopaedicSurgery,StanfordUniversitySchoolofMedicine,Stanford,CA,USA; cDepartmentof
Rheumatology,CenterofExperimentalRheumatology,UniversityHospitalZurich,Zurich,Switzerland;dBotnarResearchCenter,NIHROxford
BiomedicalResearchUnit,NuffieldDepartmentofOrthopaedics,RheumatologyandMusculoskeletalSciences,UniversityofOxford,Oxford,
UK;eStructuralGenomicsConsortium,UniversityofOxford, Oxford,UK;fMusculoskeletalResearchGroup,InstituteofCellularMedicine,
NewcastleUniversity,Newcastle-upon-Tyne,UK;gFacultyofMedicalSciences,BioinformaticsSupportUnit,NewcastleUniversity,Newcastle-
upon-Tyne,UK;hDepartmentofPhysiologyandPharmacology,SchulichSchoolofMedicineandDentistry,UniversityofWesternOntario,
London,ON,Canada
ABSTRACT ARTICLEHISTORY
Osteoarthritis(OA)isamajorclinicalproblemacrosstheworld,inpartduetothelackofdisease- Received25February2016
6 modifying drugs resulting, to a significant degree, from our incomplete understanding of the Accepted10March2016
1
0 underlying molecular mechanisms of the disease. Emerging evidence points to a role of epige- Publishedonline25April
2
y netics in the pathogenesis of OA, but research in this area is still in its early stages. In order to 2016
a
M summarize current knowledge and to facilitate the potential coordination of future research KEYWORDS
3 activities, the first international workshop on the epigenetics of OA was held in Amsterdam in DNAmethylation;
0
1 October 2015. Recent findings on DNA methylation and hydroxymethylation, histone modifica- epigenetics;histones;
4 tions, noncoding RNAs, and other epigenetic mechanisms were presented and discussed. The osteoarthritis;regulatory
08: workshop demonstrated the advantage of bringing together those working in this nascent field RNAs
at and highlights from the event are summarized in this report in the form of summaries from
y] invitedspeakersandorganizers.
sit
r
e
v
ni
U
e Osteoarthritis There are currently no disease-modifying drugs for
astl OA, with the only treatment options being analgesia,
c Osteoarthritis (OA) is a common, late-onset disease
w
physicaltherapy,weightloss,andultimatelyjointrepla-
Ne characterized by the loss of the articular cartilage of
[ cement. It is imperative therefore that our understand-
y synovialjoints.Itprincipallyaffectsthehands,thehips,
b ing of the causes of the disease is improved, and in this
d andtheknees(1).Thediseaseispresentthroughoutthe
e regardtherehasbeenatremendousamountofresearch
d world and demonstrates a greater prevalence in
a
o activity over the last decade to understand the disease
nl females.OAhasaprofound impacton patientmorbid-
w fromamolecularandcellularperspective.Mostofthese
o ity, with millions of joint replacements for OA per-
D studieshavefocusedoncartilageandthesinglecelltype
formed per year across the globe. The disease also
responsible for forming this tissue, the chondrocyte.
leads to increased mortality, resulting from secondary
A number of insights have emerged from these investi-
cardiovasculareventsarisingfromlowerpatientactivity
gations, including the role of a range of signaling path-
(2). The disease is therefore a major global health-care
ways in maintaining cartilage integrity, the shift from
problem and the burden of OA will continue to rise as
anabolism tocatabolismthat increasesdisease risk,and
average population age increases (3).
theroleofgeneticfactorsinpredisposingtoOA(5–8).
Thediseasepresentsclinicallyasachronicallypainful
OA is a particularly polygenic disease, with no risk-
joint in the absence of any overt inflammation.
conferring alleles of large singular impact. The disease
Radiographic diagnosis highlights a narrowing of the
does not transmit within families in a Mendelian fash-
joint space as the cartilage begins to focally thin. Other
ion. Instead, OA presents as a standard multifactorial,
radiographic features can include the formation of
complex trait. The mapping of OA loci has therefore
osteophytes at the margins of the joint and sclerosis of
required the use of large cohorts in case–control asso-
theunderlyingbone.Thediseasethereforeaffectsmulti-
ciation studies. These have taken the form of candidate
plejointtissues(4),butthelossofthecartilageispivotal.
CONTACTJohnLoughlin [email protected] InstituteofCellularMedicine,NewcastleUniversity,4thFloorCatherineCooksonBuilding,
FramlingtonPlace,Newcastle-upon-TyneNE24HH,UK.Tel:[phone].
©2016IngridM.Meulenbelt,NidhiBhutani,WouterdenHollander,SteffenGay,UdoOppermann,LouiseN.Reynard,AndrewJ.Skelton,DavidA.Young,FrankBeier,andJohn
Loughlin.PublishedwithlicensebyTaylor&Francis
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttribution-NonCommercial-NoDerivativesLicense(http://creativecommons.org/licenses/by-nc-nd/4.0/),
whichpermitsnon-commercialre-use,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited,andisnotaltered,transformed,orbuiltuponinanyway.
2 I.M.MEULENBELTETAL.
gene-based studies and the hypothesis-free genome- The first international workshop on the
wide approach, testing hundreds of thousands of sin- epigenetics of OA
gle-nucleotide polymorphisms (SNPs). So far up to 16
The OA epigenetics field is however still quite small
replicated OA susceptibility loci have been detected at
and, like many nascent research areas, lessons are still
or around the genome-wide significance threshold of p
< 5 × 10−8 (6,8). Functional studies using cell lines and being learnt regarding data analysis and interpretation,
and also the sample sizes needed to draw robust con-
patient tissues have revealed that effects on gene
clusions. In response to this ongoing epigenetics
expression appear to be a common mechanism by
research and the likelihood that such research will
which risk-conferring alleles mediate their OA suscept-
play an increasing role in our understanding of this
ibility; clear examples are the OA-associated SNPs
complex disease, a workshop was organized at the
rs143383, rs225014, and rs3204689 and their correla-
Dutch Royal Academy of Science in Amsterdam on
tion with expression changes at the genes GDF5, DIO2,
the 20th and 21st of October 2015. The aims of the
and ALDH1A2, respectively (9–12).
workshop were to discuss the current status of OA
epigenetics research, to provide overviews of particular
activities, to provide guidance on particular techniques
Epigenetics
(the analysis of DNA methylation arrays and of RNA
6 Epigenetics refers to a heritable change in gene expres- sequencing data), and to offer a flavor of the status of
01 sion that occurs without a change to the underlying epigenetics research in another arthritic disease, rheu-
2
y DNA sequence. There are three known mechanisms of matoid arthritis (RA). The workshop had 3 organizers
a
M epigenetic gene regulation: DNA methylation, histone (Frank Beier, John Loughlin, and Ingrid Meulenbelt), 8
3
0 modifications, and noncoding regulatory RNAs. The invited speakers, and 12 oral abstract presentations
1
4 firsttwomechanismsregulategeneexpressionbymod- selected from those who registered. In total, 60 scien-
8:
0 ulating gene transcription, whereas regulatory RNAs tists from 14 countries and 4 continents gathered to
] at act post-transcriptionally (13). Epigenetic patterns are present and to discuss their OA epigenetics research.
y
sit both plastic, especially during development and cell Reumafonds (http://www.reumafonds.nl/) and the
er differentiation when they undergo dynamic changes, Osteoarthritis Research Society International (http://
v
ni and stable, allowing cellular identity to be maintained oarsi.org/) provided financial support for the
U
e during mitotic cell division. Epigenetics provides the workshop.
astl cellwithamechanismtorespond toitsenvironmentat This article provides summaries of the presentations
c
w the cellular, tissue, and organismal level. from seven of the invited speakers (Nidhi Bhutani,
e
N
Louise Reynard, Andrew Skelton, Udo Oppermann,
[
y
b David Young, Wouter den Hollander, and Steffen
ed Epigenetics and OA Gay) and from one of the organizers (Ingrid
d
oa Meulenbelt), capturing their key points. Frank Beier
nl During the last few years, there have been a number of
w also presents a discussion on higher-order nuclear
o studies undertaken into the role of epigenetics in OA
D organization and OA. The article ends with a look to
etiology. These studies started with an investigation of
the near future of how epigenetics will enhance our
the epigenetic regulation of the expression of genes
understanding of the causes of this most common of
coding for proteins that have a known central role in
joint diseases.
the homeostasis of joint tissue, and in particular carti-
lage (14). There have also been direct investigations of
the differences in expression levels of regulatory RNAs
Stable cytosine modifications in the OA
between OA and non-OA cells, the aim being to iden-
epigenome: new kids on the block
tify key RNAs that could initiate or exacerbate disease
development (15,16). The advent of high-throughput DNA methylation on the base cytosine [5-methylcyto-
CpGDNAmethylationarrayshasenabledinvestigators sine (5mC)] was long-thought to be a stable epigenetic
to directly compare, at a genome-wide level, OA and markleadingtogenesilencing(25).Recentstudieshave
non-OA methylomes and to compare methylation sta- however revealed that DNA methylation is both
tus between DNA from different sites within the joint dynamic and reversible and have identified the key
andbetween differentjoints(17–22).Thesearrays have enzymes and pathways mediating DNA demethylation
also been used to assess whether there are direct links (reviewed in 26). Nidhi Bhutani of Stanford University
between OA genetic risk loci and epigenetic status, and presented her groups’ investigations into DNA hydro-
between gene expression and epigenetics (23,24). xymethylation on the cytosine (5hmC), which has been
EPIGENETICSOFOSTEOARTHRITIS 3
discovered to be a stable cytosine modification along- 5hmC as well as between the less stable 5fC, 5caC, and
side 5mC and which is now known to be an indepen- unmethylated cytosines (38). These tools consist of (a)
dent epigenetic mark (26,27). The enzyme family that antibody-based enrichment using specific anti-5hmC,
converts 5mC to 5hmC is the ten-eleven-translocation -5mC, -5fC, and 5caC antibodies and (b) affinity-based
(TET) family of proteins consisting of three known enrichment after specific modifications of 5hmC
members: TET1, 2, and 3 (27,28). These TET proteins (39,40), followed by high-throughput sequencing. In
are also capable of further oxidizing 5hmC to 5-for- addition, methods have been developed that can be
mylcytosine (5fC) and 5-carboxylcytosine (5caC) (29), used in conjunction with bisulfite sequencing for a
intermediates that are acted upon by base excision base resolution mapping of 5hmC (TET-assisted bisul-
repair enzymes like thymine DNA glycosylase, leading fite sequencing (41)), 5fC (5fC chemical-modification
to replacement by an unmodified cytosine and hence assistedbisulfitesequencing(41)),and5caC(5caCche-
active DNA demethylation (30,31). The precise mical-modification-assisted bisulfite sequencing (42)).
mechanisms leading to a stable 5hmC generation or Utilizing these tools, it is possible to study precise
5caC generation and subsequent demethylation are genome-wide distribution of these cytosine modifica-
however not yet clear. tionsinOAjointcellsinordertounderstandhowthese
Overtheyears,evidencehasbeenaccumulatingfora stable cytosine modifications and DNA methylation
criticalroleforDNAmethylationanddemethylationin anddemethylationdynamicsinfluencethegeneexpres-
6 OA pathogenesis (32). Alterations in DNA methylation sion changes observed in OA and other rheumatic
1
0
2 patterns have been observed in OA chondrocytes, par- diseases.
y
a ticularly a loss of DNA methylation at the promoters
M
03 and the concurrent “unsilencing” of various OA-asso- Candidate gene analysis of DNA methylation
8:41 cILia-t1eβd, geanneds iniNclOudSing(1M4,3M3P,334,).9, Ranecden1t3l,y,ADPAroMfeTssSo4r, in OA
at 0 Bhutani’s team reported a remarkable dysregulation of The application of measuring DNA methylation con-
y] 5hmC homeostasis in patients with OA, with increases tent of CpGs sites at targeted genes was covered by
sit in global 5hmC levels observed in OA chondrocytes Ingrid Meulenbelt of the Leiden University Medical
r
e
v when compared to normal chondrocytes (35). 5hmC Center. Targeted methylation of CpG sites has been
ni
U gain at specific sites in the promoters of key OA genes studied in the context of OA susceptibility genes,
e
stl was associated with increased gene expression. mainly to explore whether the risk-conferring alleles
ca Genome-wide 5hmC distribution in normal and OA that were found to influence in cis gene expression act
w
e chondrocytes identified a total of 70,591 differentially via modulation of DNA methylation of transcription-
N
y [ hydroxymethylated regions (DhMRs); 44,288 were ally active CpG sites that are close to or that are them-
b
d increased in the OA chondrocytes as compared to the selves SNPs (43,44). Such targeted analysis of
e
d normal chondrocytes and 26,303 were decreased (36). methylation levels at positional CpG sites is typically
a
o
nl Analysis of the genomic locations of these DhMRs performed by subjecting bisulfite converted DNA to
w
o revealed that the majority of 5hmC gain in OA occurs MALDI-TOF mass spectrometry (Epityper, Sequenom;
D
in gene bodies. Upon assessing the 5hmC distribution (45))ortopyrosequencing(46).Epityper issuitablefor
intheactivatedorrepressedgenes,itwasobservedthat high-throughput analysis, but cannot always capture
activated genes demonstrated elevated 5hmC in both every CpG of interest, whereas pyrosequencing is not
the promoter regions and throughout the gene body in suitableforhigh-throughputanalysisbutquantification
OA chondrocytes in comparison to normal chondro- of methylation at any CpG site is possible.
cytes, whereas the repressed genes exhibited no signifi- By applying Epityper analyses of 20 CpG sites that
cantchanges(36).Inlightoftheseobservations,further mapped across the DIO2 locus, it was shown that
studies are required on the distribution of these newly methylation at a particular CpG site 2 kb upstream of
identified cytosine modifications and their effects on the promoter enhanced DIO2 expression, particularly
the OA epigenome and transcriptome. in carriers of the OA risk C allele of the rs225014 T>C
A major caveat in widely prevalent techniques to SNP (43). Since this CpG maps within an active
assess DNA methylation, such as bisulfite conversion, CCCTC-transcription factor (CTCF) binding site,
is the failure to distinguish between 5mC and 5hmC methylation-dependent binding of this CTCF is sug-
(37). In light of the recent observations regarding a gestedtoactasapositionalisolatorofDIO2expression
genome-wide role for 5hmC alterations in OA chon- (43).Additionally,byapplyingpyrosequencinganalyses
drocytes, it is imperative to utilize recently developed of methylation at a CpG site located in the 5’ untrans-
chemical tools that can distinguish between 5mC and lated region of GDF5, it was shown that the OA risk T
4 I.M.MEULENBELTETAL.
allele of the rs143383 C>T SNP, which had previously
Detection P Val
been shown to correlate with GDF5 expression in Raw Data
Read Raw IDAT
articularcartilage(10),disruptsafunctionalCpGdinu-
PhenoTable
cleotide (44). Furthermore, the reported allele-specific
expression (ASE) of GDF5 in heterozygous carriers Normalised
Normalisation
Data
marked by the rs143383 alleles is affected by the extent
e
of methylation of the respective CpG (44). Together, pl
m
these studies demonstrated OA risk alleles that confer e Sa Probe Filtering FNiloterrmedal iDseadta
v
susceptibility to OA via changing the epigenetically o
m
modulated gene expression. Nonetheless, genome- Re
Normalised
wide analysis of the transcriptome and methylome of QC
Filtered Data
articular cartilage revealed that only 24% of the differ-
entially methylated CpG sites correlated to proximal in
Linear Model
cis gene expression and as such could be considered Fit DM Probes
transcriptionally active in articular cartilage (19,24).
Figure 1. Flow chart of the main steps in analyzing Illumina
Moreover, it was shown that for 31 and 26 OA carti-
DNA methylation array data. QC: quality control; DM: differen-
lage-relevant genes, methylation and expression,
tially methylated; Pheno: phenotype.
6 respectively, are additionally affected by genetic varia-
1
0
2 tionproximaltothesegenes,whichmayfurthermodify
y
a the OA pathophysiology (19,24).
M
3 http://bioconductor.org/packages/release/bioc/html/
41 0 Methylation microarrays: where to start wateRmelon.html, http://bioconductor.org/packages/2.
8: 8/bioc/html/methylumi.html). Once the IDAT file is
0
at Genome-wide studies of the human methylome have inputted, all packages will recommend a normalization
y] been assisted by microarray technology for a number procedure; this is a mathematical process to make sam-
sit of years, with the principal array provider being ples comparable to one another and is crucial. Probe
r
e
v Illumina with their HumanMethylation series of filtering is an essential step prior to downstream data
Uni BeadChips. The array platform has grown from 27,578 analysis.Eachprobeonthearrayhasadetectionpvalue
stle CpG dinucleotide probes (the 27K chip) covering over based on how confident the intensity call is, and probes
ca 14,000 genes to 485,000 CpG probes (the 450K chip) withpvaluesgreaterthan0.01shouldbeconsideredfor
w
e covering 99% of the Reference Sequence genes. It is removal. Additional probe filtering, for example,
N
y [ very important to consider the experimental design removal of sex chromosome probes or those containing
d b beforeconductingamethylationarray,andinthisregard SNPs, should be considered based on the experimental
e
d Louise Reynard and Andrew Skelton of Newcastle design. Leaving in probes containing SNPs with a high
a
nlo University offered guidance in their presentation. There minor allele frequency can lead to data biases, whilst
ow are biases between chips and there are positional effects gender is often an overwhelming source of variation in
D
within a chip,and soeven before datahavebeen gener- methylation studies. Quality control (QC) should be
ated, samples should be randomized within and across carriedoutonallsamplestoensurethatthedatagener-
chips to allow for correction of technical variation. A ated are as expected; the Minfi QC Report tool, or the
phenotype table should also be created, which includes MethylAid (http://bioinformatics.oxfordjournals.org/
all relevant sample information (such as gender, cell content/30/23/3435) package, will allow visualization of
type, and any treatment regimen); this table will aid potential biases in the experiment, such as bisulphite
downstream data analysis. A flow chart listing the main conversion and chip hybridization. Additional tools,
steps in processing data generated using the Illumina such as principle component analysis, are very good at
methylation arrays is shown in Figure 1. The raw data identifying known technical variation. If samples are
generated by the Illumina scanners come in a standard removed for QC reasons, then the normalization and
binary format called IDAT files, and there are many probe filtering steps must be repeated.
packages available for analyzing these files in There are two types of metric in methylation micro-
Bioconductor (https://www.bioconductor.org/), an array data, beta values, and M values. Beta values are
open-source repository of bioinformatics software between 0 and1,and areequated to percentage methy-
packages that includes Minfi, CHAMP, WateRmelon, lation.Mvalues arethelogratioofmethylationagainst
and methylumi (http://doi.org/10.1093/bioinformatics/ unmethylation intensities. Whilst beta values are useful
btu049, http://doi.org/10.1093/bioinformatics/btt684, for graphical representation and interpretation of the
EPIGENETICSOFOSTEOARTHRITIS 5
data, M values should be used for statistical analysis geneactivation.Importantly,histonemodificationsand
because of the heteroscedastic nature of beta values, DNA methylation act in concert with respect to gene
which leads to increased false positives (47). Linear regulation because both activities are functionally
models should be used for any differential methylation linked (50).
tests performed, such as the Limma package, as they At present, several classes of histone modifications
allow the flexibility of simple to complex experimental and their respective enzymatic modification systems
designs and can account for identified technical have been identified (51–53). Amongst their epigenetic
variation. substrate marks, lysine and arginine modifications are
As of October 2015, there had been four published probably the best studied: acetylation and methylation
cartilage methylome studies using the 450K array, and of lysine residues, as well as methylation of arginine.
Dr.ReynardandMr.Skeltonhavecombineddatafrom Whereas acetylation of histone tails is correlated with
three of these (Newcastle, Leiden and Utah; 19–21). geneactivation,theinfluenceofhistonemethylationon
They have a total of 179 cartilage samples composed regulating gene transcription depends on the exact
of 35 control hips, 39 preserved OA hips, 21 lesioned residue methylated and the number of added methyl
OA hips, 73 preserved OA knees, and 11 lesioned OA groups, both for arginine and lysine residues. The
knees. Using a Benjamini–Hochberg adjusted p value ≤ involvement of histone modifications in the regulation
0.01 and a Δbeta of ≥0.1, they have identified 3,424 of key aspects of the musculoskeletal system, for exam-
6 differentially methylated loci (DMLs) between control ple, in inflammation or differentiation, has recently
1
0
2 hip and OA hip cartilage, and 5,781 DMLs between been described (54). The best understood systems of
y
a preserved OA hip and preserved OA knee cartilage. histone modifications that potentially allow transmis-
M
3 These data will form the basis of an OA methylome sion of stable heritable marks through cell divisions
0
1 database that they are currently establishing, and in the comprise methylation of H3K9 (HP1, heterochromatin
4
8: future, they hope to be able to include methylome data establishment), and of H3K27 and H3K4 (repression
0
at generatedbyothergroupsfromwithinthejointbiology and activation of genes through polycomb and
y] and OA fields. trithorax complexes, respectively).
sit A chemical probe is a reagent that perturbs specifi-
r
e
v cally a protein’s function and allows the user to ask
ni Histone modifications are critical elements of
U mechanistic and phenotypic questions about its mole-
e the epigenomic landscape: using chemical tools
stl to interrogate epigenetic biology culartargetinbiochemical,cell-basedoranimalstudies
a
c (55). Chemical probes are impactful and complemen-
w
e The importance of local chromatin structure for con- tary to genetic approaches, such as CRISPR/Cas and
N
y [ trolling gene expression was the focus of the presenta- RNA interference (RNAi). They can rapidly and rever-
b
d tion by Udo Oppermann of the University of Oxford. sibly inhibit a protein function in cells or animals.
e
d Accessible chromatin, such as that found in euchroma- When coupled with RNAi, they can distinguish
a
o
nl tin, provides the environment for gene-regulatory pro- between effects due to scaffolding and effects due to
w
o teins such as transcription factors or remodeling inhibition of catalytic or protein-interaction activity.
D
complexes to interact with their cognate binding sites These features of chemical probes are relevant for
withintheregulatoryregionsofgenes,suchasproximal translational studies and target validation efforts. Such
promoters, enhancers, or silencers (48). For example, chemical tools are currently being developed for epige-
trimethylated lysine 9 of histone 3 (H3K9me3) indi- netic biology by Professor Oppermann and his collea-
cates heterochromatic or repetitive regions, whereas gues at the Structural Genomics Consortium (http://
H3K4me3 marks regulatory elements associated with www.thesgc.org/scientists/groups/oxford), an interna-
active promoters or transcription start sites and tional public–private partnership that promotes open-
H3K27me3 marks those for developmentally repressed access drug discovery and target validation
genes. Modification systems (so-called writers and era-
sers of chromatin marks) that covalently alter specific
Higher-order nuclear organization and OA
residues of chromatin proteins play a pivotal role in
this process and generate a so-called “histone code” Another emerging area in epigenetic regulation of gene
(49) that recruits specifically recognition domains and expression is the three-dimensional organization of
components (readers) of transcriptional complexes, chromatin in the nucleus, which was described by
which thus serve as the effectors of the modification. Frank Beier of the University of Western Ontario. It
In this complex and interdependent manner, chroma- is now well accepted that chromosomes and chromatin
tin-modifying systems exert control of global and local are not randomly distributed in the nucleus, but
6 I.M.MEULENBELTETAL.
organized in a highly sophisticated manner that is 200,000 isoforms are predicted to exist (64). The role
closely linked to transcriptional activity (56). Such of such regulatory RNAs in OA was the focus of the
higher-order organization includes the folding of large presentation by David Young of Newcastle University.
genomic regions, chromatin looping that connects In terms of microRNAs, miR-140 still represents the
enhancers and promoters, and interactions of chromo- most abundant, cartilage-specific, small RNA. Deletion
somes with the nuclear periphery (e.g., the lamina). ofmiR-140inmicecausesashorterskeletonandearly-
These interactions result in the formation of a number onset OA (65). MicroRNAs are transcribed as a stem-
ofdomainssuchasactivechromatinhubs,topologically looptranscriptwitha-5pand-3pmicroRNA,eitheror
associating domains (TADs), and lamin-associated both of which can be functional after processing (13).
domains (56,57). A number of key proteins mediating Because the miR-140 null mouse lacks both miR-140-
these interactions have been identified, most notably 5pandmiR-140-3p,itisunclearwhichismostactivein
the previously mentioned factor CTCF and the cohesin cartilage. Both are highly induced in a model of
complex (58). The function of these proteins in carti- mesenchymal stem cell (MSC) differentiation to chon-
lage, other joint tissues, or OA has not been addressed, drocytes; however, analysis of the regulated transcrip-
although the association of a CTCF binding site with tome leads to the prediction that miR-140-5p is the
the before-mentioned DIO2 SNP (43) points toward dominant microRNA since the transcripts that are
important roles. most downregulated are significantly enriched for pre-
6 Moreover, studies on developmental defects in dictedmiR-140-5ptargets(66).Infact,othergenesthat
1
0
2 humans and mice suggest essential functions of CTCF arealsodownregulated,butlessrobustlyso,areactually
y
a and its partners in the skeleton. For example, limb- predicted targets of a previously undefined variant, or
M
3 specific knockout mice for Ctcf completely lack fore- isomer, of miR-140-3p. This leads to the hypothesis
0
1 limbswhiletheirhindlimbsareseverelyshortened(59). that miR140-5p is a major factor in chondrogenesis,
4
8: Similarly,disruptionofCTCF-associatedTADsinmice while the isomer of miR-140-3p fine-tunes the process.
0
at and in human cells has been linked to limb malforma- For lncRNAs, RNA sequencing (RNA-seq) analysis
y] tions(60).Finally,mutationsingenesencodingcohesin of cartilage and MSC chondrogenesis has identified
sit components give rise to limb malformations (in addi- several thousand transcripts, many novel and/or differ-
r
e
v tion to other defects) in both human patients and entially expressed between normal and OA cartilage.
ni
U animal models (61). Given the close links between One such novel, cartilage-specific, noncoding RNA,
e
stl skeletal development and OA pathogenesis (62), it is termed SOX9nc2, is located upstream in the genome
a
c therefore likely that CTCF and cohesin proteins are ofthegenefortheimportantchondrocytetranscription
w
e alsoessentialfornormalcartilageandjointhomeostasis factor SOX9. Depletion of the SOX9nc2 transcript, by
N
y [ in the adult. Nevertheless, specific functions of these RNAi, prevents chondrogenesis and concomitant
d b proteinsandthree-dimensionalchromatinorganization induction of SOX9 expression. Stem cell chondrogen-
de in the adult joint and in OA have not been reported. esis models often require TGFβ and SOX9nc2 appears
a
nlo Similarly, specific approaches that have been developed to regulate the activity of TGFβ, possibly through reg-
ow to examine three-dimensional chromatin interactions, ulating the levels of TGFβ-receptor expression. There
D
such as chromatin conformation capture (3C) and its are numerous mechanisms as to how lncRNAs exert
derivatives (4C, 5C, HiC, etc.) as well as ChIA-PET their affects (67,68) and still this requires exploring for
(63), have yet to be applied to adult cartilage and SOX9nc2.
joint tissues. Overall, though chondrocytes as a cell type have
largely been overlooked by large sequencing consortia,
data show they express a wealth of noncoding tran-
Regulatory RNAs and OA
scripts (69), many of which appear novel. Future work
The completion of the human genome project revealed will be needed to investigate the function of many of
a surprising dearth of protein-coding genes, circa these in vitro and in vivo.
20,000. However, with the advent of massively parallel
sequencing technologies the complexity of the tran-
RNA sequencing
scriptome made by the genome is becoming apparent.
The transcriptome now includes 2,588 microRNAs Gene expression microarrays have been utilized in OA
(small, 22–23 nucleotide long, post-transcriptional reg- research for a number of years, having yielded valuable
ulators of gene expression), an abundance of other information on transcriptome wide gene expression
small RNAs (SnoRNAs, Y RNAs, and Pi-RNAs), and measurements of OA affected tissues such as articular
long noncoding RNAs (lncRNAs), for which over cartilage (70–74), synovium (75–77), and subchondral
EPIGENETICSOFOSTEOARTHRITIS 7
bone (78,79). The accompanying bioinformatic chal- genome. As RNA-seq is becoming more common in
lenges hereof have now been addressed and multiple transcriptome studies, currently multiple aligners sup-
easy-to-usesoftwaresolutionsarecurrentlyapparentto port alignment of so-called split reads (e.g., BWA (87),
aid in silico experiments for research groups that have TopHat2 (88), GSNAP (89), and Bowtie2 (90)), but
traditionally no background in computational biology differintheunderlyingmethod.Asidefromthealigner
(80,81). Presently, transcriptome-wide gene expression used, it is important that a proper reference genome to
measurements are increasingly being performed using align against must be chosen. Whereas general refer-
sequencing efforts, specifically RNA-seq, as opposed to ence genomes like hg19 or hg38 serve their purpose in
microarray-based experiments. RNA-seq was therefore RNA-seq experiments to estimate gene expression
the focus of the presentation by Wouter den Hollander levels, when one is interested in alternative splicing
of Leiden University Medical Center. RNA-seq offers and/or ASE it should be noted that genetic variation,
increased sensitivity (82), as well as more comprehen- and likely RNA editing even more so, considerably
siveanalysesofmechanismssuchasalternativesplicing influences whether reads will be aligned properly and
and ASE (83); however, as such it also requires less must thus be addressed (91).
convenient bioinformatic tools and might thus be less
accessible for researchers without the computational
Epigenetics of RA
know-how. Library preparation, sequencing protocol
6 and depth, read alignment, and the statistical estima- Based on the previous observation from his laboratory
1
0
2 tionofgeneexpressionlevelssubstantiallyinfluencethe that synovial fibroblasts (SFs) from patients with RA
y
a outcome of the respective experiment. While most can invade into cartilage without the stimulating influ-
M
3 bioinformatic practices that involve handling sequen- ence of immune cells and proinflammatory cytokines
0
1 cing data are compared against one another somewhat (92), Steffen Gay of University Hospital Zurich tasked
8:4 regularly (84–86), there is no golden rule on when to his group with searching for the causes of this aggres-
0
at implement what solution. Consequently, proper QC is sive behavior.
y] key to understanding whether a methodological Since his group and others could demonstrate that
sit approach serves the scientific question posed. A fre- synovial fibroblasts in RA (RASF) are not only effector
r
e
v quently used QC tool for sequencing experiments is cellsrespondingtopro-inflammatorycytokines,butact
ni
U FastQC (http://www.bioinformatics.bbsrc.ac.uk/pro endogenously when activated, they searched for the
e
stl jects/fastqc/),whichaimstoprovideaneasilyaccessible epigenetic modifications leading to the activated phe-
a
c method to inspect raw, but also processed sequencing notype of these RASF; this search could also identify
w
e data. It generates, in a modular way, web reports for a novel targets (93). As early as the year 2000, they had
N
y [ numberofstatisticssuchas,butnotlimitedto,perbase discovered that RASF are hypomethylated and thereby
b
d sequence quality, nucleotide content, sequence length endogenously activated through the upregulation of
e
d distribution, and k-mer content. Generally, upon visual matrix-degrading enzymes and potent chemokines
a
o
nl inspection of FastQC reports, sequencing data need to (94). This led his laboratory to focus on epigenetics
w
o be processed so that unique sample adapters and low- (95)includinghistoneacetylation,methylation,sumoy-
D
quality base calls among reads are removed prior to lation, and noncoding RNAs. This research was and
alignment. Again, multiple tools are available that still is supported by a number of European
achieve these objectives (e.g., cutadapt and sickle) and Commission grants with closely collaborating centers
it should thus ideally be tested what tool performs best throughout Europe, including the consortia Autocure,
in agiven experimentaldesign (e.g.,single-end sequen- Masterswitch, Osteoimmune, TEAM, and BeTheCure.
cingcomparedtopaired-endsequencing).Whenoneis His group’s most recent research has focused on the
certain that the reads are of ample quality to continue, analysis of methylation of novel transcription factor
an aligner must be chosen to explore what regions of genes. Thereby they have shown that the promoter of
the genome are found to what extent in the mRNA TBX5 is hypomethylated in RASF and induces the
pool, reflecting gene expression levels. As opposed to activation of these cells to produce proinflammatory
aligning DNA sequencing data that can theoretically cytokines and chemokines including IL8, CXCL12,
cover regions across the entire genome, RNA-seq data and CCL20 (96). His colleague Michel Neidhart has
inherentlyonlyconsistofexonreads.Asaconsequence further analyzed polyamine metabolism in RASF and
of that, read aligners must be aware of the fact that a developed a novel therapeutic strategy to inhibit one of
substantial number of reads from an RNA-seq experi- the key enzymes regulating methylation (97).
ment might consist of multiple exons, which are by Since RASF are modified in their state of acetylation
definition not directly adjacent to one another on the through the expression of specific bromodomains
8 I.M.MEULENBELTETAL.
(acetyl lysine binding domains), another colleague of fromthegeneticstudiesistheneedtostudylargesample
Professor Gay, Kerstin Klein, has studied these bromo- sizes, although the tens of thousands needed in GWAS
domains, which are involved in chromatin remodeling are unlikely to be necessary for epigenetic studies, with
and transcriptional activation. She has discovered that several hundred well-phenotyped samples being a more
BRD2-4 could be inhibited with BET-inhibitors, which realistictargetnumberatthisstage.Althoughthemajor-
are currently of interest to a number of global pharma- ityofOAepigeneticsresearchhasbeencartilage-centric,
ceutical companies (98). other relevant joint tissues are being investigated (107)
A detailed analysis by two further colleagues of and this joint-wide approach will be of great value in
Professor Gay, Caroline Ospelt and Mojca Frank- elucidatingbothtissue-specificandjoint-wideepigenetic
Bertoncelj, of microRNAs and lncRNAs revealed differ- effects. With regard to pain, which is the number one
encesinthetranscriptomeofSFsthatisdictatedbyjoint- complaint of OA patients, epigenetic changes in the
specific positional differences in the origin of the cells. nervous system should also be considered, especially
This could be a contributing factor to RA and was pre- since the chronic pain in these patients suggests an epi-
sentedasaplenarylectureatthe2014annualmeetingof geneticcomponent.
theAmericanCollegeofRheumatology(99).Acomplete Whilst a tremendous amount therefore remains to be
analysis of microRNA profiling in RASF versus normal done, the workshop did highlight a number of advances
SFs has been undertaken by his group and the role of andwasagreatsuccessinbringingscientistsandtrainees
6 specific microRNAs further identified. For example, withinterestsinthisareatogethertodiscussfutureplans.
1
0
2 miR-155 is expressed at a higher level in monocytes The requirement to share resources, knowledge, and
y
a than in RASF (100) and has a pivotal role in collagen- expertisewasastrikinglyclearconclusionandhasalready
M
3 induced arthritis (101). Moreover, his group demon- led to the very tangible outcome of the OA methylome
0
1 strated that miR-203 is regulating IL-6 (102) and that database that was discussed earlier. It is hoped that this
4
8: miR-18a enhances not only the IL-6-mediated produc- OA epigenetics workshop will be the first of many; a
0
at tionoftheacute-phaseproteinsfibrinogenandhaptoglo- desire for future workshops will be a mark of advance-
y] bininhumanhepatocytes (103),butalso,wheninduced ment inthis important OA research field.
sit byTNFα,activatesRASFthroughafeedbackloopinNF-
r
e
v κB signaling (104). His group has also shown that the
ni
U targeting of microRNAs by antago-miRs can be highly Acknowledgments
e
stl effective(105)with,forexample,miR-20ainhibitedbyan
a Wewouldliketothankallofthosewhomadethisworkshop
c antago-miRforthetreatmentofpulmonaryhypertension
w possible, in particular the abstract presenters and all other
e (106). Last but not least, his group has also shown that
N delegateswhoensuredthatwehadascientificallystimulating
y [ miR-323 regulates TIMP3 and thereby inhibits the acti- meeting.
b
d vation of the highly potent proinflammatory cytokines
de TNFαandIL-6,providinganovelstrategytoinhibitboth
a
o
nl the cytokine-dependent and the cytokine-independent Declaration of interest
w
o pathways ofRA.
D We thank Reumafonds (http://www.reumafonds.nl/) and the
Osteoarthritis Research Society International (http://oarsi.
Conclusions and perspectives org/) for their generous encouragement and their financial
support of speaker expenses. The authors report no conflicts
As shown in numerous talks at the workshop, there is ofinterest.Theauthorsaloneareresponsibleforthecontent
verystrongevidenceforakeyroleofepigeneticmechan- and writing of the article.
isms in the pathogenesis of OA. This raises hopes that
drugs targeted at specific epigenetic regulators (many of
whicharecurrentlyunderdevelopmentforthetreatment References
of cancer and other diseases) might be useful in the
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treatment of OA. However, research in this area is still 2006;332:639–42.
atanearlystage,withmechanisticlinksbetweenspecific 2. HawkerGA,CroxfordR,BiermanAS,HarveyPJ,Ravi
epigenetic changes and disease progression still unclear B, Stanaitis I, Lipscombe LL. All-cause mortality and
in many cases. As was observed in the analysis of OA serious cardiovascular events in people with hip and
knee osteoarthritis: a population based cohort study.
genetics, the stratification of OA phenotypes by, for
PLoS One 2014;9:e91286.
example, severity of disease, site of disease, and the
3. Hunter DJ, Schofield D, Callander E. The individual
involvement of other clinical parameters may aid the and socioeconomic impact of osteoarthritis. Nat Rev
identificationofepigeneticmarkers.Anadditionallesson Rheumatol 2014;10:437–41.